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Reversible Acyl-Homoserine Lactone Binding to Purified Vibrio fischeri LuxR Protein. M. L. Urbanowski, 2004.The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors . Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon . We have purified LuxR from recombinant Escherichia coli . Purified LuxR binds specifically and with high affinity to DNA containing a lux box . This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex . When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E . coli RNA polymerase to initiate transcription from the luxI promoter . Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent . Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different . The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density . Conservation of Plasmid Maintenance Functions between Linear and Circular Plasmids in Borrelia burgdorferi. Philip E. Stewart, 2003.The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection . Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified . Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B . burgdorferi . These vectors identify internal regions of linear plasmids necessary for autonomous replication in B . burgdorferi . Although derived from linear plasmids, the vectors are maintained in circular form in B . burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form . Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication . In Vitro Antifungal Activities of Inhibitors of Phospholipases from the Fungal Pathogen Cryptococcus neoformans. Ranjini Ganendren, 2004.Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein . These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA) . Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy . We determined in C . neoformans var . grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated . Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes . Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors . These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D) . The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test . Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A2 at 0.25 µM . Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal . We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal . They also provide tools for assessing the relative importance of the various enzyme activities in virulence . Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets . Genome-Wide Transcriptional Profiling in a Histidine Kinase Mutant of Helicobacter pylori Identifies Members of a Regulon. Mark H. Forsyth, 2002.To identify putative members of a regulon controlled by the H . pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H . pylori strains and analyzed bacterial gene transcription using DNA arrays . Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains . Strain-dependent variations in differential expression were also detected . These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H . pylori . Characterization of a New Operon, as-48EFGH, from the as-48 Gene Cluster Involved in Immunity to Enterocin AS-48. Marta Diaz, 2003.Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2 . A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52) . In this region, the as-48A structural gene and as-48B, as-48C, as-48C1, as-48D, and as-48D1 genes and open reading frame 6 (ORF6) and ORF7 had been identified . The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression . Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level . Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid . The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection . On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed . This cluster of genes is expressed by two polycistronic mRNAs, T2 and T3, in JH2-2(pAM401-81) in coordinate expression . Our results also suggest that expression of T3 could be regulated, because in JH2-2(pAM401EH) transformants, T3 was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D1 gene for immunity against AS-48 .
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